Studies On Changes Of Physiology And Biochemistry In The Course Of Shoot Regeneration Of Mustard

In this research, the plant regeneration system were established on three varieties of mustard (including Sichuanzhacai, Jinyanyihaoxiangcai and Yuandatoucai). In the process of the shoots were regenerated from cotyledons, this work mainly focused on the research of biochemical indicator changes (soluble protein, peroxidase, superoxide dismutase, catalase, soluble sugars, starch and nucleic acid), soluble proteins and peroxidse isoenzyme of the three varieties of mustard. The results were listed as follows:1. The most suitable medium to get the highest shoot regeneration frequency was the MS medium containing 6-BA3.0mg/L and NAAO.lmg/L, and the frequencies (Sichuanzhacai, Jinyanyihaoxiangcai and Yuandatoucai) were 83%,76% and 51%.2. The optimal medium for callus induction of Jinyanyihaoxiangcai was MS+O.1mg/LNA A+3.0mg/L6-BA+0.1mg/L2,4-D, and the induction rate was 93%. The optimal for callus prol ifelation was MS+NAA0.3mg/L+6-BA0.3mg/L+0.1mg/L2,4-D. The optimal formula for callus differentiation was MS+0.6mg/L6-BA+0.1mg/L NAA, the rate is 13%.3. In the process of the shoot regeneration from cotyledons of three mustard varieties, the changes of antioxidant enzymes (POD, SOD and CAT) activity, soluble sugar content, starch content and RNA content had the similar tendency. In the differentiation process, there were a litte difference during three varieties. The shoots regeneration of Sichuanzhacai was earlier than the other two varieties and its biochemical change was appeared the peak earlier. The differentiation rate of Sichuanzhacai and Jinyanyihaoxiangcai were higher than Yuandatoucai, and their physiological and biochemical indexes changed intenser than Yuandatoucai. Jinyanyihaoxiangcai formed adventitious bud without callus in the same medium, the soluble protein content and DNA content showed different trends.4. The protein and POD isoenzymes components in different development stages of three mustard varieties were compared by SDS-PAGE. There were different specific proteins and POD isoenzymes appeared and disappeared in different stages.There were four kinds of proteins (55KDa,30KDa,31 KDa and 18KDa) expressed during callus of Sichuanzhacai differentiated, and four kinds of proteins (57KDa,38KDa,28Kda and 12KDa) disappeared. When cotyledons of Jinyanyihaoxiangcai differentiated, two kinds of proteins (55Kda and 14KDa) expressed and four kinds of proteins (57KDa,34KDa,26Kda and 12KDa) didnt express. When cotyledons of Yuandatoucai cultured 16 days, the callus differentiated, the protein of 55KDa expressed and protein of 12KDa disappeared. The protein of 12KDa didnt express during three varieties of mustard organogenesis, therefore, the protein of 12KD disappeared can be used as a marker of cotyledons differentiation in mustard tissue culture.The cotyledons of three mustard varieties had the same POD isoenzymes. The cotyledon of Sichuanzhacai differentiated, while B band disappeared and the intensity of C, D and E bands then enhanced. Jinyanyihaoxiangcai cotyledon differentiation occurred when the A band appeared. When cotyledon of Yuandatoucai differentiated, A and C bands arised. They all have A, B, C, D and E bands.5. The principal component analysis showed that the antioxidant enzymes (POD, SOD and CAT) activity, soluble sugar content, soluble protein content and DNA content could reflect the process of the shoot regeneration from mustard cotyledons.